The present invention is directed to new reagents for the qualitative and quantitative measurement of ligands such as antigens and haptens in biological fluids. The reagents are comprised of at least ten dye molecules or at least one dye polymer having at least ten dye monomers per polymer bonded to an antibody through an isothiocyanate group on the dye.
Dye molecules have been used as labels for proteins (e.g. antibodies) to make reagents which have been primarily associated with fluorescence readings. It is generally accepted that labeling an antibody with a dye molecule and measuring the absorbance value does not provide a level of sensitivity sufficient to enable an accurate quantitative measurement of ligands in immunoassay systems. This is because there are obstacles to obtaining a sufficiently sensitive reagent whose change in absorbance values can be accurately measured by standard equipment such as a spectrophotometer or by the naked eye. (See "Labeling of Proteins with Fluorescent Dyes" J. J. Haaijman, Immunohistochemistry Edited by A. C. Cuello pp. 47-85, 1983)
To obtain a sufficiently sensitive dye-labeled antibody reagent, it is necessary to create reagents having a sufficient number of dyes bound to each antibody to provide maximum absorbance.
Dye molecules which have a relatively high molar extinction coefficient value (.epsilon.) increase sensitivity. Dye molecules satisfy this requirement are known in the art and include isothiocyanate derivatives of aminofluorescein and aminophthalocyanines as well as Chicago Sky Blue and Evans Blue. The molar extinction coefficient value (.epsilon.) is a measure of the optical density of the dye at a one molar concentration measured at the maximum absorbance peak. The greater the number of optical density units per molar solution, the higher the level of sensitivity of the dye.
The art has not been successful in producing a reagent having a sufficient number of dyes bound to each antibody to provide a sensitive reagent for immunoassay testing.
Prior to the present invention it was believed that the number of dyes which could be bound to an antibody must be limited so as to avoid having the dyes interfere with the binding sites on the antibody for binding to the ligand to be detected.
Highly over-conjugated antibodies tend to decrease antibody activity which makes quantitative measurement difficult. Therefore, the art has limited the number of dyes bound to a protein (e.g. antibodies) (see Immunohistochemisty p. 56 showing four dye molecules bound to a protein; and U.S. Pat. No. 4,261,968, column 25, lines 35-40 showing a maximum of nine dye molecules per antibody molecule).
However, such prior art reagents are not sensitive enough for use in immunoassay procedures for the quantitative measurements of ligands, especially tests on serum samples containing low concentrations of antigens and haptens and for visual tests without the aid of absorbance measuring equipment.
It is therefore an object of the present invention to provide a sensitive reagent for use in immunoassay procedures wherein the reagent comprises at least ten dye molecules or at least one dye polymer having at least ten dye monomers per polymer bound to an antibody through an isothiocyanate group on the dye molecule or polymer.
It is another object of the invention to provide methods of making such reagents.
It is a further object of the invention to employ these reagents in immunoassay tests for the qualitative and quantitative detection of ligands in biological fluids.
It is a still further object of the invention to provide immunoassay procedures for qualitatively and quantitatively detecting the presence of Apoprotein A-1 in serum samples.